This application is a Phase II STTR to further the study of a novel therapeutic intervention for ischemic stroke. Phase I investigated the mechanism of protection against ischemic stroke provided by the novel designer therapy, recombinant T cell receptor ligand (RTL), a selective modulator of the inflammatory function of myelin- reactive T cell specificities. Our findings suggest that RTL protects against ischemia-induced brain injury only if the RTL contains a neuroantigen peptide and the matched Class II MHC moiety of the recipient. We demonstrate that RTL containing mouse MHC coupled to mouse myelin peptide is effective in reducing ischemic damage in mice (RTL551). Similarly, RTL1000 is a human MHC coupled to a human myelin peptide. RTL1000 is not effective in wild-type mice, but provides significant protection in 'humanized' HLA-DR2 mice that selectively express only this human MHC class II allele. As such, this treatment represents a new therapy targeting inflammatory immune cell populations specifically triggered by brain injury. This post-ischemic therapeutic approach is promising because it specifically suppresses brain targeted immune responses while leaving other portions of the immune system intact. The inflammatory response after ischemia in the brain has been studied extensively and modulation of this inflammatory response (immunotherapy) improves outcome in experimental models. However, these therapies have not translated successfully to patients in clinical trials. There is now support for the idea that stroke is a multi-organ systemic disease with interactions between the peripheral immune system and the injured and recovering brain. Many patients who survive the initial injury to the brain suffer CIDS, CNS injury-induced immunosuppression, resulting in fatal infection. Therefore, immunotherapy targeted towards minimizing brain damage following ischemia must minimize CNS inflammation while at the same time not exacerbating CIDS. This is a significant hurdle that our group is well poised to study. Our group has pioneered the study of cerebral ischemia-induced changes to peripheral immune organs using the middle cerebral artery occlusion (MCAO) mouse model of ischemic stroke. This application will test three specific hypotheses 1) that RTL1000 has an extended therapeutic window and 2) that RTL1000 retains its protective potential in middle aged and old aged mice. Finally, 3) that RTL1000 is an effective therapy when combined with tissue plasminogen activator (tPA). Successful completion of the current proposal will determine the efficacy of RTL1000 in treatment of ischemic stroke, and its ability to be used in combination with tPA, ultimately providing a roadmap for designing pilot clinical trials. Upon completion of this Phase II STTR proposal, in partnership with Virogenomics, we will proceed to a Phase I safety trial in Stroke patients. PUBLIC HEALTH RELEVANCE: Stroke is one of the leading causes of death and the leading cause of disability in the United States. Unfortunately, there is currently only a single drug available to improve outcome following ischemic stroke, tissue plasminogen activator (tPA), with a limited therapeutic window of >4.5 hrs. The proposed studies will demonstrate the efficacy of RTL1000 in treatment of ischemic stroke, and its ability to be used in combination with tPA.